Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops

Li, Feiwu; Wang, Yan; Long, Likun; Xing, Qi; Li, Cong-Cong; Zhang, Shihong

doi:10.3390/ijms150915109

Cited by 30 publications

(11 citation statements)

References 35 publications

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“…Second, in the LAMP method, four to six primers are used to synthesize target DNA amplicons. If six primers are used amplification time is reduced, but the probability of cross-contamination by aerosols is increased (Li et al, 2014;Notomi et al, 2000). When four primers are used, however, the amplification time required for detection of the result takes more than an hour (Kang et al, 2015;Kikuchi et al, 2009;Leal et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

A new on‐site detection method for Bursaphelenchus xylophilus in infected pine trees

et al. 2019

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The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease (PWD), which is a major problem in East Asia and West Europe. Quick identification of PWN is needed to prevent the dispersal of PWD to healthy forests. Various detection methods of PWN have been developed using anatomical characters and molecular markers. These methods are not suitable for rapid diagnosis because it is difficult to distinguish B. xylophilus from the non‐pathogenic species Bursaphelenchus mucronatus based on morphological characters without expertise in nematode taxonomy and most PCR or isothermal amplification detection methods require time‐consuming processes. In this study, we developed an on‐site PWN detection method using a recombinase polymerase amplification (RPA) assay with a novel extraction buffer (DAP buffer). This new PWN detection method is able to extract genomic DNA from PWN in pinewood by simple buffer consisting of sodium hydrate, polyethylene glycol 200 and dimethyl sulfoxide in 10 min without using the experimental devices and able to distinguish between B. xylophilus and other Bursaphelenchus spp. by amplifying the species‐specific 5S rDNA fragment of B. xylophilus in 10 min. Taken together, our protocol can obtain the result for the detection of PWN in pine tree samples within 30 min. This result suggests that RPA/DAP assay is much faster, easier and cheaper than the conventional methods for detecting PWN.

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Section: Discussionmentioning

confidence: 99%

A new on‐site detection method for Bursaphelenchus xylophilus in infected pine trees

et al. 2019

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“…In addition, cry2Ab and cry3A genes, two of the most important insect-resistant exogenous genes, have been detected by visual LAMP within 60 min at an isothermal condition of 63˚C. And the sensitivity of LAMP assay was five copies of haploid genomic DNA, about five-fold greater than that of classic PCR assays [26].…”

Section: The Screening Of Gmomentioning

confidence: 95%

Loop-Mediated Isothermal Amplification (LAMP) Assay for GMO on: Recent Progresses and Future Perspectives

Li¹,

Ji²,

Zhao³

et al. 2015

OALib

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As more and more genetically modified (GM) crops are approved for commercialization and planting, safety issues of GM crops have become hot topics worldwide. For both regulatory and academic purposes, development of rapid, economic and effective on-site detection methods for GM components is indispensible. Up to now, the most effective and sensitive techniques used for GMO detection are based on polymerase chain reaction (PCR). PCR method needs expensive, heavy instruments and gel electrophoresis. Therefore, it is commonly used in laboratory test, and unsuitable for on-site detection. Loop-mediated isothermal amplification (LAMP), an isothermal nucleic acid amplification technique, has been extensively used in many areas such as food safety and clinic diagnosis. Advantageous characteristics of LAMP, such as high specificity and sensitivity, simple operation, low cost, eye visualization, particularly free of special equipment, render it with high potential to be used for GMO on-site detection. In this review, we summarized current status of the application of LAMP in GMO detection, and discussed possible improvements needed for its adaptability regarding to on-site GMO detection. Hopefully, the information present here would facilitate the practical risk assessment of GMO.

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“…To date, however, most of the studies focused on the analysis of Cry1 toxins, and few have been targeted to Cry2A. Li, Yan, et al (2014) developed a LAMP assay for the determination of Cry2Ab, the limit of detection was five copies of haploid genomic DNA. established a MAb based DAS-ELISA for the analysis of Cry2Ab, with a LOD of 1 pg/g.…”

Section: Phage-displayed Nanobodies Based Das-cliamentioning

confidence: 99%

Phage-displayed nanobody based double antibody sandwich chemiluminescent immunoassay for the detection of Cry2A toxin in cereals

Qiu

Liu

et al. 2019

Food and Agricultural Immunology

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In this study, we developed a double antibody sandwich chemiluminescent immunoassay (DAS-CLIA) based on phagedisplayed nanobodies for the determination of Cry2A toxin. Nine phage-displayed nanobodies specifically to Cry2A were obtained from a naive nanobody library after four rounds of biopanning. The phage-displayed nanobody P2 with high affinity binding was selected as the detection antibody for the sensitive DAS-CLIA development. The proposed method exhibited high sensitivity with a limit of detection of 0.09 ng/mL, has a wide linear range for Cry2A toxin detection, in the range of 0.1-1000 ng/mL and negligible cross-reactivities to the other Bt toxins. The recoveries of Cry2A toxin spiked in cereal samples (rice, wheat, and corn) ranged from 82.7% to 118%, with a coefficient of variation of less than 10%. This study demonstrated that the DAS-CLIA procedure based on phage-displayed nanobodies may provide an alternative sensitive method for the detection of Bt toxins in agri-products.

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Development and Application of Loop-Mediated Isothermal Amplification Assays for Rapid Visual Detection of cry2Ab and cry3A Genes in Genetically-Modified Crops

Cited by 30 publications

References 35 publications

A new on‐site detection method for Bursaphelenchus xylophilus in infected pine trees

A new on‐site detection method for Bursaphelenchus xylophilus in infected pine trees

Loop-Mediated Isothermal Amplification (LAMP) Assay for GMO on: Recent Progresses and Future Perspectives

Phage-displayed nanobody based double antibody sandwich chemiluminescent immunoassay for the detection of Cry2A toxin in cereals

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